Masafumi Fukushima

Synthetic analogues of vitamin D3 with an oxygen atom in the side chain skeleton A trial of the development of vitamin D compounds which exhibit potent differentiation‐inducing activity without inducing hypercalcemia

Four analogues of vitamin D3 with an oxygen atom in the side chain skeleton were synthesized to determine whether their differentiation‐inducing activity could be separated structurally from their activity to induce hypercalcemia. The order of the in vitro potency to reduce nitroblue tetrazolium in human myeloid leukemia cells (HL‐60) was 22‐oxa‐1α25‐(OH)2D3 > 1α,25‐(OH)2D3 > 20‐oxa‐1α,25‐(OH)2D3≒22‐oxa‐1α‐(OH) D3 > 1α‐(OH)D3 > 20‐oxa‐1α‐(OH)D3. 22‐Oxa‐1α,25‐(OH)2D3, was also about 10‐times more potent than 1α,25‐(OH)2D3 in suppressing proliferation and inducing differentiation of mouse myelomonocytic leukemia cells (WEHI‐3), but the former was much weaker than the latter in inducing the release of 45Ca from prelabeled fetal mouse calvaria. These results suggest that the differentiation‐inducing activity of vitamin D compounds can be separated structurally from their activity to induce hypercalcemia.

Quantitative enzyme immunoassay for human granulocyte colony stimulating factor (G-CSF)

Human granulocyte colony stimulating factor (G-CSF) was detected at the pM level by a simple sequential sandwich enzyme immunoassay. The polyclonal antiserum obtained by immunizing rabbits with recombinant G-CSF did not recognize other colony stimulating factors. The EIA developed gave satisfactory reproducibility as judged by an intra-assay precision of 4.3-6.2% and an interassay precision of 6.2-9.0%. Results from this assay procedure correlated well with those of a bioassay. The method could be performed within 6 h.

Metabolism of 1α-hydroxyvitamin D3 to 1α,25-dihydroxyvitamin D3 in perfused rat liver

Abstract The metabolism of 1α-hydroxyvitamin D3 (1α-OH-D3) was studied in rat liver perfused with [3H]-1α-OH-D3. [3H]-1α-OH-D3 was converted very rapidly to a more polar metabolite, which was identified as 1α,25-dihydroxy-vitamin D3 [1α,25-(OH)2-D3] by co-chromatography with synthetic 1α,25-(OH)2-D3 as well as by gas chromatography-mass spectrometry. [3H]-1α,25-(OH)2-D3 appeared in the perfusate as early as 20 min after addition of [3H]-1α-OH-D3, and its level in the perfusate increased linearly for at least 120 min. These data strongly indicate that 1α-OH-D3 is metabolized to 1α,25-(OH)2-D3, which exerts biological effects on bone and intestine.

Radioimmunoassay for erythropoietin using anti-recombinant erythropoietin antibody with high affinity.

A sensitive radioimmunoassay (RIA) for the detection of erythropoietin (EPO) was developed using anti-recombinant EPO antibody with high affinity. The sensitivity was 100 amol/tube (5 mIU/ml) and it was possible to detect a serum EPO level between 5 and 200 mIU/ml. This method enabled us to measure native EPO as well as recombinant EPO. With this method we determined serum EPO levels in healthy individuals and patients with chronic renal disease, rheumatoid arthritis and iron deficiency anemia. Values in patients with chronic renal disease were lower than those in healthy individuals, while values in patients with rheumatoid arthritis, or iron deficiency anemia were significantly higher than those in healthy individuals.

25-hydroxylation of 1α-hydroxyvitamin D3 in vivo and in perfused rat liver

The demonstration that la-hydroxyvitamin D3 (la-OH-Ds) can replace the metabolically active form of vitamin Ds, lcy,25dihydroxyvitamin Ds (la,25(OH)z-Ds) in the treatment of various vitamin D refractory syndromes [l-3] has prompted investigations on the metabolism of this analog. In 1975, we synthesized [2-3H]-1~-OH-D3 and demonstrated that if was metabolized very rapidly to [3H]-lar,25-(OH)zD3 in perfused rat liver [4]. [3H]-1a,25-(OH)Z-D3 was found to be the only metabolite of [3H]-lcu-OHD3 produced by the liver [4]. Almost simultaneously, Holick et al. synthesized [6-3H]-la-OH-D3 and reached the same conclusion from in vivo studies in rats [5] . This paper reports in vivo and liver perfusion studies showing that 25-hydroxylation of l&OH-D3 by the liver is not under metabolic control.

Sensitive method for determination of nicorandil in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A sensitive method for the determination of nicorandil, an anti-anginal drug, has been developed. The method involves solid-phase extraction of the drug and an internal standard using a Bond-Elut PH extraction column, liquid-liquid extraction with methyl acetate and dichloromethane, and reversed-phase high-performance liquid chromatography on a mu Bondapak C18 column with an ultraviolet detector. The limit of determination in plasma was 3 ng/ml and the intra-day coefficient of variation (n = 7) was less than 10%.

History of Vitamin D Treatment of Renal Osteodystrophy

Vitamin D treatment was tried when renal osteodystrophy was first recognized in the early 20th century, using vitamin D2, D3, or dihydrotachysterol. Large doses of vitamin D2 or D3 (150,000-500,000 IU) were prescribed by monitoring serum calcium, phosphate, and alkaline phosphatase. After the discovery of 1,25-dihydroxycholecalciferol, this compound or 1 alpha-hydroxycholecalciferol was applied to the treatment of renal osteodystrophy. In a preclinical study, especially of 1 alpha-hydroxycholecalciferol, nephritogenoside nephritis was the most responsive condition. These active vitamin D preparations are now widely used in patients with chronic renal failure under hemodialysis. Other active vitamin D compounds, such as hexafluoro-1,25-dihydroxycholecalciferol and 22-oxacalcitriol, are also under investigation.

Synthetic studies of vitamin D3 analogues from bile acids. Part 3. Syntheses of 1α,25-, 1α,24R-, and 1α,24S-dihydroxycholecalciferols from lithocholic acid and their biological activities

Syntheses of 1α,25-, 1α,24R-, and 1α,24S-dihydroxycholecalciferols from lithocholic acid via the analogous 1,4,6-trien-3-ones by application of Kanekos procedure are described. A new synthesis of 1α,25-dihydroxycholecalciferol from lithocholic acid via the 1,4-dien-3-one is also reported. The biological activities of the three cholecalciferols were tested.

Synthesis of 1α-[2-3H]Hydroxyvitamin D3

1α-[2-3H]Hydroxyvitamin D3 was synthesized chemically. The preparation was radiochemically pure and had a high enough specific activity (4.2 Ci/mmol) to permit experiments using 62.5 pmol/rat. This preparation had as much effect as synthetic 1α-hydroxyvitamin D3 in increasing the serum Ca level of vitamin D-deficient rats.

Metabolism of oxyfedrine

SummaryPharmacokinetic studies with orally administered 14C-oxyfedrine were carried out in rats and also in men. 1.In the rat tissue concentrations of oxyfedrine (determined by UV absorption) and 14C-radioactivity reached a maximum within 15 min. Recovery of oxyfedrine equaled that of 14C-activity (14C-oxyfedrine + 14C-labelled metabolites) in the heart and lung tissues, but differed somewhat in the liver and kidney extracts.2.In experiments with bile fistula rats, 3 metabolites were recovered in the bile: a main metabolite, as an unlabeled, ninhydrin-positive substance (M1) besides two 14C-labeled minor metabolites*.3.Approximately 40 to 50% of the oxyfedrine applied appeared in the urine as metabolites of oxyfedrine, mainly norephedrine, in rats and men.4.The pharmacological significance of these findings is discussed.